Synthesis, α-mannosidase inhibition studies and molecular modeling of 1,4-imino-ᴅ-lyxitols and their C-5-altered N-arylalkyl derivatives

A synthesis of 1,4-imino-ᴅ-lyxitols and their N-arylalkyl derivatives altered at C-5 is reported. Their inhibitory activity and selectivity toward four GH38 α-mannosidases (two Golgi types: GMIIb from Drosophila melanogaster and AMAN-2 from Caenorhabditis elegans, and two lysosomal types: LManII from Drosophila melanogaster and JBMan from Canavalia ensiformis) were investigated. 6-Deoxy-DIM was found to be the most potent inhibitor of AMAN-2 (Ki = 0.19 μM), whose amino acid sequence and 3D structure of the active site are almost identical to the human α-mannosidase II (GMII). Although 6-deoxy-DIM was 3.5 times more potent toward AMAN-2 than DIM, their selectivity profiles were almost the same. N-Arylalkylation of 6-deoxy-DIM resulted only in a partial improvement as the selectivity was enhanced at the expense of potency. Structural and physicochemical properties of the corresponding inhibitor:enzyme complexes were analyzed by molecular modeling.


S4
and EtOAc (40 mL). Layers were separated and the aqueous layer was extracted with EtOAc (40 mL). The combined organic extracts were dried over Na 2 SO 4 , filtered and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (EtOAc/hexane 1:6) to afford pyrrolidine 6 as a colorless oil (530 mg, 83%). R F = 0.29 (EtOAc/hexane 1:6); [α]  In a manner similar to [2], 20% HCl (1.5 mL) was added to a stirred solution of acetonide 6 (64 mg, 0.26 mmol) in MeOH (3 mL) while cooling to 0 °C (ice-water bath). After 15 min of stirring, the ice-water bath was removed and the reaction mixture was stirred at rt overnight. Next, HCl was carefully neutralized with solid Na 2 CO 3 (0.6 g). The resulting suspension was filtered, the filtration cake was washed with MeOH (5 mL) and the filtrate was concentrated. The residue was suspended in CH 2 Cl 2 (10 mL), filtered and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (MeOH/CHCl 3 1:10 In a manner similar to [2], a suspension of 10% Pd-C (25 mg, 10 wt %) and pyrrolidine 6 (250 mg, 1.01 mmol) in MeOH (10 mL) was stirred at rt under a hydrogen atmosphere (balloon) for 48 h. The catalyst was filtered off, the filtrate was cooled to 0 °C (ice-water bath) and conc. HCl (0.38 mL) was added. The ice-water bath was removed and the reaction mixture was stirred at 40 °C for 2 h. The solvents were evaporated to dryness to give the corresponding hydrochloride as white solid (174 mg), which was used in next reaction without further purification and characterization. To a suspension of the hydrochloride in anhydrous DMF (10 mL), K 2 CO 3 (418 mg, 3.03 mmol, 3 equiv) and 4-iodobenzyl bromide (359 mg, 1.21 mmol, 1.2 equiv) were added and the reaction mixture was stirred at rt overnight. Next, the reaction mixture was partitioned between water (40 mL) and EtOAc (40 mL), the organic layer was separated and washed with water (40 mL). The organic layer was dried over Na 2 SO 4 , filtered and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (MeOH/CHCl 3 1:10 containing 0.5% (v/v) conc. NH 3 ) to afford pyrrolidine 8 as an In a manner similar to [2], a suspension of 10% Pd-C (6.5 mg, 10 wt %) and pyrrolidine 6 (65 mg, 0.26 mmol) in MeOH (5 mL) was stirred at rt under a hydrogen atmosphere (balloon) for 48 h. The catalyst was filtered off, the filtrate was cooled to 0 °C (ice-water bath) and conc. HCl (0.10 mL) was added. The ice-water bath was removed and the reaction mixture was stirred at 40 °C for 2 h. The solvents were evaporated to dryness to give the corresponding hydrochloride as white solid (37.7 mg), which was used in next reaction without further purification and characterization. To a suspension of the hydrochloride in anhydrous DMF (2 mL), K 2 CO 3 (102 mg, 0.74 mmol, 3 equiv) and 2-(bromomethyl)naphthalene (65 mg, 0.29 mmol, 1.2 equiv) were added and the reaction mixture was stirred at rt overnight. Next, the reaction mixture was partitioned between water (20 mL) and EtOAc (20 mL), the organic layer was separated and washed with water (20 mL). The organic layer was dried over Na 2 SO 4 , filtered and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (CHCl 3 /MeOH 10:1 containing 0.5% (v/v) conc. NH 3 ) to afford pyrrolidine 9 as a yellow oil (47.4 mg, 70%). R F 0.16 (CHCl 3 /MeOH 10 :1 containing 0.5 % (v/v) conc.
HCl (0.15 mL) was added. The ice-water bath was removed and the reaction mixture was stirred at 40 °C for 2 h. The solvents were evaporated and the product was

(3aS,4R,6aR)-Benzyl 2,2-dimethyl-4-((trityloxy)methyl)dihydro-3aH-[1,3]dioxolo[4,5-c]pyrrol-5(4H)carboxylate (11)
In a manner similar to [2], a suspension of 10% Pd-C (0.5 g, 10 wt %) and Nbenzylpyrrolidine 3 (5.06 g, 10.0 mmol) in MeOH (10 mL) was stirred at rt under a hydrogen atmosphere (balloon) for 48 h. The catalyst was filtered off and the solvent was evaporated under reduced pressure. The residue was dissolved in CH 2 Cl 2 (70 mL) and Et 3 N (3.48 mL, 25.0 mmol) was added to the reaction mixture. While cooling to 0 °C (ice-water bath), CbzCl (45% in toluene, 7.50 mL, 0.02 mol) was added to the reaction mixture. After 15 min of stirring, the ice-water bath was removed and the reaction mixture was stirred at rt for 2 h. Next, the reaction mixture was washed with water (2 × 70 mL), the layers separated and the aqueous layer was extracted with CH 2 Cl 2 (50 mL). The combined organic extracts were dried over Na 2 SO 4 , filtered and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel

General method for alkylation (Method A)
In a manner similar to [2], a suspension of 10% Pd-C (10 wt % of 13) and alcohol 13 (0.35 mmol, 1 equiv) in MeOH (5 mL) was stirred at rt under a hydrogen atmosphere (balloon) for 2 h. The catalyst was filtered off and the solvent was evaporated under reduced pressure. The crude amine was dissolved in anhydrous DMF (5 mL/0.35mmol of amine) and the reaction mixture was cooled to 0 °C (ice-water bath). K 2 CO 3 (1.4 equiv) and the corresponding bromide (1 equiv) were added and the reaction mixture was stirred for 15 min. Then, the icewater bath was removed and the reaction mixture was stirred at rt overnight. The reaction mixture was partitioned between water (50 mL) and EtOAc (25 mL) and the layers were separated. The organic layer was washed with water (50 mL) and brine (5 mL), dried over Na 2 SO 4 , filtered and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel.

General procedure for deprotection (Method B)
In a manner similar to [2], 20% HCl (1 mL) was added to a stirred solution of protected acetonide (0.15 mmol) in MeOH (2 mL) while cooling to 0 °C (ice-water bath). After 15 min of stirring, the ice-water bath was removed and the reaction mixture was stirred at rt for 72 h. Next, HCl was carefully neutralized with solid Na 2 CO 3 (950 mg, 1.5 equiv to HCl). The resulting suspension was filtered, the filtration cake was washed with MeOH (5 mL) and the filtrate was concentrated. The residue was suspended in DCM (5 mL), filtered and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography.

]dioxolo[4,5-c]pyrrol-4-yl)ethanol (22)
In a manner similar to [2], a 10% NaOH solution (5 mL) was added to a solution of carbamate 21 (289 mg, 1.36 mmol) in EtOH (25 mL) and the reaction mixture was refluxed for 24 h. The solvent was evaporated under reduced pressure and the residue was partitioned between brine (30 mL) and CHCl 3 (30 mL). The layers were separated and the aqueous layer was extracted with CHCl 3 (30 mL). The combined organic extracts were dried over Na 2 SO 4 , filtered and the solvent was evaporated under reduced pressure. The

General method for alkylation (Method C)
In a manner similar to [2], K 2 CO 3 (1.7 equiv) was added to a stirred solution of pyrrolidine 22 (1 eq.) in anhydrous DMF (5 mL/0.46 mmol of 22) and the reaction mixture was cooled to 0 °C (ice-water bath). The corresponding bromide was added (1.3 equiv) and the reaction mixture was stirred for 15 min. The ice-water bath was removed and the reaction mixture was stirred at rt overnight. Then, the reaction mixture was partitioned between water (10 mL) and EtOAc (10 mL), the layers were separated and the aqueous layer was extracted with EtOAc (5 mL).
The combined organic extracts were washed with water (3 × 10 mL), dried over Na 2 SO 4 , filtered and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography.

Enzyme assay
Recombinant soluble forms of Drosophila melanogaster Golgi (GMIIb) and lysosomal (LManII) α-mannosidases as well as Caenorhabditis elegans Golgi α-mannosidase AMAN-2 were produced in Pichia pastoris and enriched by ammonium sulfate precipitation or nickel chelation chromatography as previously described [3,4]. The αmannosidase from Canavalia ensiformis (JBMan) was purchased from Sigma. The mannosidase activity of these enzyme preparations were measured using p-nitrophenyl α-D-mannopyranoside (pNP-Man; Sigma; 100 mM stock in dimethylsulfoxide) as a substrate at 2 mM final concentration in 50 mM acetate buffer of the relevant previously defined optimal pH, GMIIb and AMAN-2 at pH 6.0, LManII at pH 5.2, and JBMan at pH 5.0) and 0.5 μL of the enzyme (0.05 µg of protein for JBMan), in a total volume of 50 μL for 1-2 h at 37 °C. GMIIb was assayed in the presence of 0.5 mM CoCl 2 .
The lyophilized derivatives used in the assay were dissolved in DMSO to the final concentration 50 mM and further diluted to a desired concentration in water. These derivatives were preincubated with the enzyme in the buffer for 5 min at rt and the reaction was started by addition of the substrate. The reactions were terminated with two volumes (0.1 mL) of 0.5 M sodium carbonate and the production of p-nitrophenol was measured at 405 nm using a multimode reader Mithras LB943 (Berthold Technologies). The average or representative result of three independent experiments made in duplicate is presented. The IC 50 values were determined with 2 mM pNP-Man.
The K i values were determined from Dixon plots of assays performed with pNP-Man (0.5-4 mM). S16 consisted of more than 30 amino acid residues, Zn 2+ ion and the bound inhibitor were built using the Facio program [26]. The hybrid orbital projection operator (HOP) technique was used in the generation of fragments for the covalently bounded amino acids. The FMO calculations were performed using the second-order Møller-Plesset theory [27,28] (MP2) with the 6-31G(d) basis and polarizable continuum model (PCM) [29]. The Gamess package [30,31] [version 30 June 2021 (R1)] was used. The virtue of the FMO technique is to predict pair interactions between the two structural fragments of the molecular system embedded within the electrostatic potential of the surroundings (IFIEinter fragment interaction energy). FMO-PIEDA enables the separation of the interaction energy into physically interpretable The electrostatic energy E els originates from Coulomb-like interactions between the fragments, the exchange energy E exch arises for fermion particles, the electrons, and accounts for the Pauli repulsion of electrons between the fragments. E ct+mix is somewhat peculiar; it includes the charge transfer that results from electron transfer from occupied molecular orbitals of one fragment to the vacant virtual orbitals on the second fragment. The mixing part is basically an approximate polarization. Dispersion energy E disp originates from interactions of instantaneous fluctuations of dipoles on the fragments due to electron correlation. This method was recently used to analyze interaction energy in different biomolecular systems [32][33][34][35][36][37].
To understand an inhibitory effect of a substituent at C-5 of the inhibitor ring of 10, 20, 28, 29, 30, 31 and DIM, the inhibitor was divided into two fragments, the pyrrolidine ring structure (I ring ) and the methyl moiety (I linker ) (in